The long-term goal of the proposed studies is to determine the physiological role(s) of the regulatory light chains of myosin (RLC) in the regulation and/or modulation of skeletal muscle contraction-The central hypothesis is that the RLC play an important role in the regulation and/or modulation of striated muscle contraction. Specifically, the hypotheses to be tested are that 1) Ca2+ and/or Mg+ binding to the single Ca2+-Mg2+ binding site on the RLC and 2) the phosphorylation of Ser (by Ca2+- calmodulin activated myosin light chain kinase, MLCK) play important roles in the regulation and/or modulation of contraction. In the first three years of this grant considerable progress has been made in understanding the role of the RLC in contraction. We have shown that a) the RLC affect crossbridge cycling; b) phosphorylation of the RLC increases the Ca2+- sensitivity of both force development and thin filament activated myosin ATPase activity; c) phosphorylation of the RLC increases maximal force production and d) the Ca2+-Mg2+ binding site is required for the phosphorylation induced shift in the Ca2+- sensitivity of force development. The latter suggests that there is coupling between the Ca2+-Mg2+ binding site and the phosphorylation site. In addition, we have shown that the level of endogenous RLC phosphorylation, in addition to the role played by Troponin, is a crucial determinant of the Ca2+- sensitivity of force development in skeletal muscle, the magnitude of which was not previously appreciated. Although these in vitro results have told us much about the function of the RLC, it is still not totally clear what their in vivo function is. It is also true that the role of the RLC in skeletal muscle has been fraught with controversy and much of this has come from the fact that methods to study the function of the RLC have not been available. Unfortunately there have been no artifact-free methods for the selective extraction/replacement of the RLC in either myosin or in more complex systems, e.g., myofibrils and skinned muscle fibers and this has contributed to the controversy. A more unequivocal way of approaching the role of the RLC would be to develop systems whereby the RLC can be manipulated in a native setting. Several powerful approaches are available today that make this possible and these include transgenic and knock-in/out mouse models. To test the above hypotheses, transgenic and knock-in/out animal models, where various mutants of RLC will replace the endogenous mouse RLC, will be utilized. Sophisticated physiological studies on both intact muscle and skinned fibers from these animals will be performed to determine the role of the RLC in skeletal muscle contraction and regulation. The proposed studies will allow us to uniquely study the role of the RLC in striated muscle contraction and to determine their in vivo role.